1. Volume 47, Issue 5, Pages 669-680 (September 2012)
Noncanonical Mismatch Repair as a Source of Genomic Instability in Human Cells 
Javier Peña-Diaz, Stephanie Bregenhorn, Medini Ghodgaonkar, Cindy Follonier, Mariela Artola-Borán, Dennis Castor, Massimo Lopes, Alessandro A. Sartori, Josef Jiricny 
Molecular Cell 
Volume 47, Issue 5, Pages (September 2012)
DOI: /j.molcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Figure 1 Noncanonical MMR of Uracil Promotes PCNA-Ubn
(A) Efficiency of U/G→C/G repair in a U/G-U/G substrate in nuclear extracts of BL2 cells as assessed by the intensity of the 1,516 and 1,307 bp AclI restriction fragments (see text and Figure S1 for details). Where indicated (+), the extracts were supplemented with aphidicolin (Aphi), the UNG2 inhibitor Ugi, or both. (In this and subsequent figures, panels with a dark background show a UV-shadowing image of a 1% agarose gel stained with GelRed.)
(B) Autoradiograph of the gel shown in (A).
(C) Schematic representation of the substrate used to detect strand bias of the repair synthesis, showing the position of the BssSI restriction sites and the Nt.BstNBI nicking site.
(D) Denaturing agarose gel electrophoresis of the U/G-U/G substrate digested with BssSI and Nt.BstNBI, after incubation with Ugi (lane 1), BL2 extracts (lane 2), or both (lane 3). M, size marker.
(E) Autoradiograph of the gel shown in (D).
(F) Efficiency of U/G→C/G repair in a U/G-U/G substrate incubated with extracts of BL2 cells, supplemented (+) with Ugi and/or purified recombinant MutLα, either wild-type (WT) or mutated in the endonuclease active site of PMS2 (mut).
(G) Autoradiogram of the gel shown in (F).
(H) Immunoblot of reactions shown in (F). PCNA-Ub, monoubiquitylated form of PCNA. N.I., normalized incorporation; N.L., normalized levels.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Noncanonical MMR in Mammalian Cell Extracts Promotes PCNA-Ubn Independently of the Type of Lesion
(A) Efficiency of repair of the U/G-U/G, U/G, G/T, or meG/C substrates in nuclear extracts of 293T-Lα cells, either MLH1 deficient (–) or proficient (+).
(B) Autoradiogram of the gel shown in (A).
(C) Immunoblot of reactions shown in (A). β-Tub, β-tubulin loading control. N.I., normalized incorporation; N.L., normalized levels.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 PCNA-Ubn In Vitro Correlates with Gap Formation
(A) AclI digest of the U/G-U/G substrate or of control plasmid (C/G-C/G), either covalently closed or containing the indicated gaps, after incubation with BL2 nuclear extracts. Size of single-stranded region (in nucleotides) is shown below the pictograms.
(B) Autoradiogram of the gel shown in (A).
(C) Immunoblot of reactions shown in (A).
(D) AclI digest of the U/G-U/G substrate recovered from reactions containing dialyzed nuclear extracts of 293T-Lα cells expressing (+) or lacking (−) MLH1 and treated with Ugi, which were supplemented with increasing concentrations of the four dNTPs.
(E) Autoradiograph of the gel shown in (D).
(F) Immunoblot of reactions shown in (D). β-Tub, β-tubulin loading control.
(G) Quantification and size distribution of gaps detected in a U/G-U/G substrate recovered after incubation with a BL2 extract. The substrate was linearized as described in Supplemental Information. Substrate incubated with heat-inactivated extract was used as control. n > 400.
(H) Electron micrograph of a representative gap. Arrows indicate the ssDNA region in the magnified inset (upper right) and in its graphic representation (lower right). N.I., normalized incorporation; N.L., normalized levels; ∗∗p < 0.01; n.s., not significant.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Activation of MMR In Vivo Induces PCNA-Ubn and pol-η Recruitment to Chromatin
(A) Scheme of siRNA transfections and MNNG treatment of 293 cells.
(B) Immunoblot of total extracts of cells in which MLH1, PMS2, or MSH6 was knocked down by siRNA transfection 2 days prior to treatment with 2 μM MNNG. The extracts were analyzed 0, 24, and 48 hr after MNNG treatment. Luc, luciferase siRNA.
(C) Immunoblot of total- and chromatin-bound proteins in HCT116 (MMR-deficient) and HCT116+Chr3 (MMR-proficient) cells treated with 5 μM MMNG for 3 hr prior to harvesting or mock treated. β-Tub, β-tubulin loading control. N.L., normalized levels.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Noncanonical MMR Can Be Activated in the G1 Phase of the Cell Cycle
(A) FACS profiles of exponentially growing (upper panels) or serum-starved (lower panels) MMR-proficient (HCT116+Chr3) and -deficient (HCT116) cells treated with 5 μM MNNG.
(B) Immunoblot analysis of the above cells 24 or 48 hr after MNNG treatment.
(C) Immunoblot of the chromatin-bound protein fractions in the above cells at the indicated time points.
(D) Two-dimensional FACS profile of a confluent cell culture, untreated or treated for 3 hr with 5 μM MNNG in the presence of EdU. The gate was set to include cells with 2n DNA content. Similar FACS profiles were obtained with serum-starved cells.
(E) Quantitation of the EdU-positive cells with 2n DNA content corresponding to G1 cells gated in (D). The histogram represents the normalized values of the triplicate experiment and shows a clear increase in EdU incorporation in MNNG-treated MMR-proficient cells (continuous blue line). EdU incorporation is shown in arbitrary units. N.L., normalized levels.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 MMR-Induced PCNA-Ubn Is Dependent on Both Rad18 and USP1
(A) Immunoblot analysis of extracts of 293 cells treated first with siRNA against luciferase (Luc) or RAD18 and 2 days later with 2 μM MNNG (+). Extracts were made 3, 9, and 27 hr after MNNG treatment, as indicated.
(B) As in (A), but the cells were treated with siRNA against luciferase or USP1. N.L., normalized levels.
(C) Effect of RAD18 or USP1 knockdown on MNNG sensitivity of A2780-MLH1-1 (MMR+) and A2780MNU-Cl1 (MMR−) cells. The data represent the mean ± SD of three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 MMR Promotes Mutagenesis
(A) MNNG-induced mutation frequencies in A2780-MLH1-1 (MMR+) and A2780MNU-Cl1 (MMR−) cells. MNNG concentrations are shown on the abscissa.
(B) Mutation frequencies induced by 500 nM MNNG in A2780-MLH1-1 (MMR+) and A2780MNU-Cl1 (MMR−) cells pretreated with siRNAs against luciferase (Ctrl), RAD18, or USP1. The MNNG-induced mutation frequencies were obtained by subtracting the spontaneous mutation frequencies of these cell lines at the HPRT locus from the total mutation frequencies. The data represent the mean ± SEM of at least three independent experiments (see also Figure S7B). ∗∗∗p < 0.001; n.s., not significant.
(C) Putative scheme of MMR function during different phases of the cell cycle. Lesions recognized by MMR factors outside of S phase might activate ncMMR. In the absence of strand discontinuities that direct canonical MMR, nicks introduced into the DNA by the endonuclease activity of MutLα might be used for loading of EXO1. This would result in the generation of long single-stranded gaps, but this process would not necessarily lead to the removal of the lesion, given that the noncanonical activation of MutLα lacks strand bias. These gaps might persist for some time, due to low nucleotide concentrations and low levels of replicative polymerases, and their persistence might trigger PCNA ubiquitylation and recruitment of error-prone polymerase(s) such as pol-η. Unrepaired gaps would be converted into DSBs during replication and would trigger a S/G2 arrest. In contrast, lesions generated during S phase would be repaired with high fidelity, due to the existence of free termini that direct MMR to the nascent strand, the ready availability of dNTPs, and higher concentrations of replicative polymerases.
Molecular Cell  , DOI: ( /j.molcel )
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